PROJECT SUMMARY/ABSTRACT Nutrients and metabolites can both positively and negatively regulate cell signaling, anabolic metabolism, and proliferation to impact cancer growth. One nutrient that promotes cancer growth is asparagine, an amino acid cancer cells can either take up from the microenvironment or synthesize through asparagine synthetase (ASNS) from glutamine and aspartate. Cancer cell asparagine consumption is already a therapeutic target: L- asparaginase, which digests extracellular asparagine, is a standard-of-care treatment for acute lymphoblastic leukemia. Not surprisingly, leukemic resistance to asparaginase is associated with increased ASNS activity for biosynthesis. We recently reported that intracellular asparagine levels are limiting for cancer cell proliferation and that asparagine is used by cancer cells as an amino acid exchange factor for uptake of amino acids from the microenvironment. This exchange function of asparagine enables mTORC1 activation and downstream promotion of anabolic metabolism. Our preliminary data further suggest that mTORC1, which increases glycolysis along with anabolic metabolism, may be subject to feedback inhibition by lactate, the end product of glycolysis. Lactate is exported in large amounts from cancer cells through monocarboxylate transporters (MCTs), and blocking lactate export reduces tumor growth. Why do cancer cells export so much lactate? Could lactate provide a negative feedback signal to inhibit further anabolism in homeostatic situations? Do cancer cells, which generally express relatively high levels of MCTs, evade this negative feedback mechanism through lactate export? This proposal will investigate mechanisms by which growth-promoting signaling pathways are both positively and negatively regulated by metabolites, and explore ways to therapeutically target this regulation using rational combinations of existing clinical compounds. Specifically, we will: (1) Determine whether respiration supports cancer cell proliferation through asparagine production; (2) Assess rational combination treatment strategies with L-asparaginase to exploit cancer cell dependence on asparagine for growth; and (3) Examine lactate regulation of mTORC1 and activating transcription factor 4 activities. Elucidating nutrient regulation of cancer growth will promote development of better cancer treatment strategies that block tumor growth with limited opportunity for resistance.